Steroids from the Soft Coral Sinularia crassa

Soft corals have proven to be a rich source of terpenoids [1]. We previously have isolated a
series of bioactive cembrane- [24] and norcembrane- [58] diterpenoids from the Formosan
soft corals of the genus Sinularia. Previous chemical investigations on the soft coral Sinularia
crassa have led to the isolation of structurally unique steroids and cembranoids, from which
some have been shown to exhibit anti-inflammatory [9,10] and 5-reductase inhibitory
activities [11], respectively. Our continuing chemical investigation on S. crassa has led to the
isolation of one new sterol, crassarosterol A (1), and four new steroidal glycosides,
crassarosterosides AD (25) (Figure 1). The structures of 15 have been established by
extensive spectroscopic analysis, including 2D NMR (1H1H COSY, HMQC, HMBC, and
NOESY), chemical methods, and HPLC. The effect of 14 on the expression of the pro-
inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in
lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells and the cytotoxicity of
compounds 15 against a panel of cancer cell lines including human liver carcinoma (HepG2 and
HepG3), human breast carcinoma (MCF-7 and MDA-MB-231), and human lung carcinoma (A
549) were evaluated in order to discover bioactive natural products

The absolute configuration of sterol 1 has been established by Moshers method in the present
work. On the basis of biogenesis, the steroidal moieties of the glycosides 25 should possess the
same absolute configurations as shown in the formulae. Cytotoxicity of steroids 15 against
HepG2, HepG3, MCF-7, MDA-MB-231, and A-549 cancer cell lines was evaluated. The
results showed that 1 exhibited cytotoxicity toward HepG2 cancer cell line with an IC50 value of
14.9 M, while 4 also showed cytotoxicity toward HepG2 and HepG3 cell lines with IC50
values of 17.6 and 18.9 M, espectively. The other compounds were found to be inactive (IC50
> 20 M) toward the above cancer cell lines after 72 h exposure. The effect of steroids 14 on
the expression of pro-inflammatory iNOS and COX-2 proteins in RAW264.7 macrophage cells
stimulated with LPS was also evaluated using immunoblot analysis. At a concentration of 10 M
, steroid 2 was found to significantly reduce the level of iNOS protein to 12.9 4.3% and 4
could reduce the iNOS espression to 50.1 6.3%. However, these compounds could not
effectively reduce the level of COX-2. On the contrary, 13 were found to stimulate the
expression of COX-2
The melting point was determined using a Fisher-Johns melting point apparatus. Optical
rotations were determined with a JASCO P1020 digital polarimeter. IR spectrum was obtained
on a JASCO FT/IR-4100 spectrophotometer. The NMR spectra were recorded on a Varian 400
MR NMR or Varian Unity INOVA 500 FT-NMR instrument at 400 or 500 MHz for 1 H
(referenced to TMS, H 0.00 ppm for CDCl3) and 100 or 125 MHz for 13 C (referenced to C
77.0 for CDCl3). ESIMS and HRESIMS were recorded by ESI FT-MS on a Bruker APEX II
mass spectrometer. Silica gel 60 (Merck, 230400 mesh) and LiChroprep RP-18 (Merck, 4063
m) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60
F254, 0.25 mm) and precoated RP-18 F254S plates (Merck, 1.05560) were used for TLC
analyses. High-performance liquid chromatography (HPLC) was performed on a Hitachi L-7100
pump equipped with a Hitachi L-7400 UV detector at 210 nm and a semi-preparative reversed-
phase column (Merck, Hibar Purospher RP-18e, 5 m, 250 10 mm).

Animal Material
The soft coral Sinularia crassa was collected by hand using scuba off the coast of Sansiantai,
Taitung county, Taiwan, in July 2008, at a depth of 10 m, and was stored in a freezer. This soft
coral was identified by one of the authors (C.-F.D.). A voucher specimen (specimen no. SST-03)
was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen
University. Extraction and IsolationThe frozen bodies of S. crassa (1.1 kg fresh wt) were minced
and extracted with EtOH (3 2 L). The organic extract was concentrated to an
aqueoussuspension and was further partitioned between EtOAc and H2O. The EtOAc extract
(17.0 g) was fractionated by open column chromatography on silica gel using n-hexaneEtOAc
and EtOAcMeOH mixtures of increasing polarity to yield 32 fractions. Fractions 25, eluting
with EtOAcMeOH (8:1), was further separated by silica gel column chromatography with
gradient elution (n-hexaneacetone, 8:1 to 2:1) to yield four subfractions (25A25D).
Subfraction 25B was subjected to RP-18 column chromatography (MeOHH2O, gradient,

5090%), and subsequently purified by RP-18 HPLC (CH3CNH2O, 65%) to obtain compounds
1 (6.6 mg) and 5 (1.2 mg). Compound 4 (1.8 mg) was obtained from subfraction 25C using RP-
18 HPLC (CH3CNH2O, 65%). In the same manner, Subfraction 25D was fractionated over RP-
18 gel using

Prior investigation of the genus Sinularia reported on several steroidal glycosides, however, all
of these were found to possess a 24-methylene substituted side chain [22,23]. 5 is the first
example of steroidal glycoside with a 23,24-dimethyl substitited side chain from soft coral of the
genus Sinularia. Our biological data reveal that 2-O-acetyl-L-fucose functionality plays an
important role toward the inhibition of the pro-inflammatory iNOS. Steroidal glycoside 2 could
be a useful anti-inflammatory agent, while steroids 1 and 4 have shown inhibitory activity
toward the selected human liver cancer cells.